B-cell Activation Factor, BAFF (also known as BLyS, TALL-1, THANK, zTNF4 and TNFSF13B) and APRIL (also known as TRDL-1 alpha) are members of the TNF super family (TNFSF) of proteins. The prototype of the family, Tumor Necrosis Factor Alpha (TNFA), originally discovered for its in vivo effect causing tumors to regress, is a key mediator of inflammation. BAFF and APRIL proteins participate in a variety of cellular and intracellular signaling processes and are synthesized as a type 2 membrane proteins and fold into conserved β-pleated sheet structures. It is known that BAFF and APRIL form homotrimers typical of the TNFSF. In addition, BAFF and APRIL also form heterotrimers together.
The receptors for TNFSF members also represent a family of structurally related molecules, including at least 26 receptors and/or receptor decoy molecules. The extracellular domains of members of this family are composed of multiple repeats of a cysteine-rich domain (CRD), a small protein domain containing six conserved cysteines that form three disulfide bonds. The intracellular domains of these receptors are more diverse, although many members of the family contain a death domain that mediates apoptosis and other receptor signaling events. These members are all capable of inducing apoptosis via interaction with one or more intracellular adaptor molecules that also contain death domains. Other signaling receptors of this family signal via interactions with a family of adaptor molecules called TRAFs (TNF receptor associated factors). Signaling through BAFF receptors (for example, BAFFR, TACI and BMCA) is triggered by binding of an oligomeric (and for the most part, trimeric) BAFF ligand.
Recent reports have presented contradictory hypotheses regarding the functionally relevant oligomerization state of BAFF. As a member of the TNFSF of ligands, which are usually active as homotrimers, BAFF is hypothesized to function as a trimer and several reports support this hypothesis (See Kanakaraj, P. et al. Cytokine, 12, 25-31 (2001); Karpusas, M. et al. J. Mol.Biol., 315, 1145-1154 (2002); Oren, D. A. et al. Nature Struct. Biol., 9, 288-292 (2002); and Zhukovsky, E. et al., Nature, 427, 413 (2004)). However, X-ray crystallography and size exclusion chromatography (SEC) results recently reported (Liu, Y, et al. Nature, 423, 49-56, (2003); Liu, Y, et al., Nature, 427, 413 (2004) and Kim, H. M. et al. Nature Struct. Biol., 10, 342-348 (2003)) indicate that BAFF forms 60-mer structures resembling the viral capsid of Satellite Tobacco Necrosis Virus (Liu, Y. et al. Cell, 108, 383-394 (2002)). A strong pH dependence of oligomerization state was observed: at pH 6.0 BAFF was 100% trimeric and at pH 7.4 100% 60-mer. It has been proposed that the higher order species represented the active form of BAFF and that the unusually long D-E loop facilitates formation of oligomers. These higher order species would represent novel cytokine architecture. Comparison of BAFF protein constructs reported in the literature reveals that various affinity tags have been used. Previous reports showed that fusion tags may affect oligomerization state of host proteins (Wu, J. and Filutowicz, M. Acta Biochim Pol. 46, 591-599 (1999).) All references cited herein are expressely incorporated in their entirety.
The three-dimensional structures of BAFF and APRIL are made up of a sandwich of two anti-parallel beta-sheets with the “jelly roll” or Greek key topology and it assembles into a trimeric complex. The cognate receptors of BAFF and APRIL are part of the related TNFSF of receptors. Furthermore, there appears to be significant conservation of the mode of receptor binding. In general, each receptor monomer binds within the cleft formed between two of the ligand monomers. The overall similarity in tertiary and quaternary structures of both the ligands and their complexes with receptors indicates that well-proven strategies for inhibition or agonism of one ligand-receptor system may be transferable to the other proteins in the family. Thus the present invention provides methods for the creation of variants of BAFF that are modulators of BAFF, APRIL and/or TNFSF signaling pathways.
Lymphocyte populations are regulated by replication and death. B-cell maturation relies on signals through both BCR (B-cell receptor) and survival co-stimulation by BAFF. Therefore, BAFF is important for survival of B-cells and humoral immune response. Normally, only a small number of B-cells mature due to a vigorous selection directed at selecting of a population of B-cells that are not auto reactive. Overexpression of BAFF in transgenic (Tg) animals results in the state of immune hyperreactivity, which is manifested in an increase of peripheral B-cell lymphocyte counts, breakdown of B-cell tolerance (inappropriate survival of autoreactive lymphocytes), and enlarged lymphoid organs and spleen. All this is accompanied by the appearance of anti-DNA antibodies, an increase in antibody secretion (e.g., IgM, IgG and IgA), and Ig-deposition in the kidneys resulting in glomerulonephritis that leads to autoimmune-like syndromes similar to systemic lupus erythematosus (SLE), Sjogren syndrome (SS), and the like. It has also been observed that SLE, RA, and SS patients showed a correlation of high BAFF concentration with elevated levels of anti-dsDNA Ab, a biochemical marker of these diseases. It has been shown that in RA patients, concentration of BAFF in synovial fluids is much higher than in blood.
BAFF also stimulates T-cells to a much lesser degree and increases the population of activated effector T-cells. There are three known receptors of BAFF: BAFF-R, TACI, and BCMA. The first one is exclusively specific to BAFF and the latter two are shared with APRIL, another member of TNFSF and the closest homologue of BAFF. Phenotypes of BAFF knockout mice (KO) and BAFF-R mutation strain of mice (ANVySnJ) suggest that BAFF-R is the main receptor for BAFF and is responsible for control of B-cell maturation. TACI controls B-cell homeostasis and T-cell Independent immune response and appears to act as an inhibitory BAFF receptor. The role of BCMA is unclear thus far.
The therapeutic interest in BAFF as a drug target lies in its strict specificity so that attenuation of the function of this cytokine may result in no widespread side effects. Animal studies have shown that administration of BAFF-blocking reagents in BAFF Tg mice reduced diseases incidence and its severity pointing to this cytokine as a crucial mediator of theses autoimmune diseases. For example, TACI-Fc inhibited the development of proteinuria and prolonged survival of NZB/WF1 mice (the main SLE model in mice).
APRIL is expressed in the same type of cells as BAFF, that is peripheral blood lymphocytes (PBL) and monocytes/macrophages. Similar to BAFF, APRIL also co-stimulates (together with BCR) B-cell proliferation and IgM production. APRIL KO mice die in utero suggesting that it may play an important role in development. BCMA preferentially binds to APRIL over BAFF.
APRIL stimulates colon cancer cells that express no TACI or BCMA (i.e. neither of the known APRIL receptors). In a similar assay, BAFF has no effect on tumor cells. Also, s-BCMA, which can bind and block APRIL, inhibited cancer cell growth. All these facts taken together suggest the existence of a specific APRIL-R that has not been identified yet.
A need still exists for proteins that can interfere with intracellular signaling processes. Thus, it is an object of the present invention to provide proteins comprising BAFF variants with BAFF receptor-interaction domains that are modified such that each domain has significantly reduced affinity and/or signaling capacity for the one or more cognate receptor(s). Such modified domains preferably retain association with individual monomer domains of naturally occurring BAFF or APRIL, but exhibit a dominant-negative phenotype, antagonizing the action of related naturally occurring domains via their sequestration into inactive oligomeric complexes. In another embodiment, BAFF homotrimers or homodimers may bind to one or more cognate receptors, including but not limited to BAFF-R, BCMA and TACI, and inactivate or reduce one or more intracellular signaling processes. In a further embodiment, BAFF variant homo-oligomers may act as agonists or superantagonists of one or more cognate receptors.